This invention relates to a method for genetically engineering coniferous plants. In particular, this invention relates to a particle-mediated gene transfer method for producing and developing transgenic embryos for plants of the genus Pinus and Pinus interspecies hybrids. This method is well suited for producing transgenic clonal planting stock useful for reforestation.
The identification of gene function coupled with the ability to precisely manipulate DNA has enabled the construction of synthetic genes which, when properly transferred and incorporated into a host cell, can modify the cell""s genetic makeup. This capacity to manipulate genes using recombinant DNA technology combined with in vitro methods for plant propagation now permits genetic engineering of crop species. Indeed, genetic engineering processes have been used to successfully transfer foreign genes into certain plant species, thereby resulting in the recipient species acquiring a useful genetic trait (such as resistance to herbicides or insects).
The transfer of foreign genetic material into the chromosomes of a recipient plant is typically carried out through the use of either an Agrobacterium-mediated or a particle-mediated transformation process. Agrobacterium gene transfer employs the natural ability of the soil-borne bacterium Agrobacterium tumefaciens to transfer a portion of DNA (known as T-DNA) from an extrachromosomal plasmid (known as the Ti-plasmid) to a receptive plant host cell under specific conditions. Using suitable techniques of recombinant DNA manipulation, the T-DNA may be replaced with a desired piece of DNA. This method has not proven suitable for all plant cell types.
In particle-mediated gene transfer, the DNA of interest is precipitated onto the surface of carrier particles which are subsequently accelerated toward a piece of target tissue. The carrier particles penetrate the cell wall of the plant cell, wherein the DNA can be expressed, and may integrate with the chromosomal DNA. Transient expression of the transforming DNA has been reported in conifers (Charest et al., 1993; Walter et al., 1994). Stable expression only results if the transforming DNA integrates with the chromosomal DNA.
In addition to Agrobacterium-mediated and particle-mediated gene transfer, other methods of gene transfer have been used to introduce foreign genes into conifers, such as electroporation (Campbell et al., 1992). However, only transient expression has been reported using any of these other methods, and no transgenic plants have been reported to have been generated using such methods.
This illustrates that the mere act of introducing DNA into the host cell chromosome is, by itself, not sufficient for the production of transgenic plants. A tissue culture system that enables the multiplication and subsequent development of the transformed cells is also an important component of a successful genetic transformation protocol.
A method known as somatic embryogenesis is sometimes employed in the clonal propagation of certain conifers. Propagation by somatic embryogenesis refers to methods whereby embryos are produced in vitro from small pieces of plant tissue or individual cells. The embryos are referred to as somatic because they are derived secondarily, from somatic (vegetative) tissue, rather than directly from the sexual process. Vegetative propagation via somatic embryogenesis has the capability to capture the genetic gain of highly desirable genotypes. Furthermore, these methods may be readily amenable to automation and mechanization to produce large numbers of plants of individual clones (e.g. for reforestation purposes).
It was not until 1985 that somatic embryogenesis was demonstrated in conifers and the first viable plantlets were regenerated from conifer somatic embryos. Since 1985, conifer tissue culture workers throughout the world have pursued the development of somatic embryogenesis systems capable of regenerating plants. The goal of much of this work is to develop conifer somatic embryogenesis as an efficient propagation system for producing clonal planting stock en masse.
The two most economically important conifer genera are Picea (spruce) and Pinus (pine). Those working in conifer somatic embryogenesis have found that there is a striking difference between Picea conifers and Pinus conifers as to the ease with which somatic embryogenesis can be initiated and plants regenerated. In fact, when one measures the respective levels of achievement in the art of conifer somatic embryogenesis among species of these two important genera, it is clear that significantly more success has been obtained with Picea than with Pinus. Indeed, the recalcitrance of embryogenic cultures of Pinus species is well documented. This is especially true for pines commonly found in the southeastern United States (known in the industry as Southern yellow pines).
Nevertheless, researchers working with Pinus species plants have recently achieved some important advances. For example, U.S. Pat. Nos. 5,413,930, 5,491,090, 5,506,136, 5,677,185, 5,731,191, 5,731,203, and 5,731,204 (which are hereby incorporated by reference) disclose multi-step methods that are able to complete the entire somatic embryogenesis regenerative process, from explant collection to planting of somatic embryo derived plants, for historically recalcitrant Southern yellow pines (i.e., Pinus taeda, Pinus serotina, Pinus palustris, and Pinus elliottii), Pinus rigida, and hybrids thereof
Scientists have found the dichotomy exhibited by Picea conifers and Pinus conifers in the area of somatic embryogenesis also exists in the field of genetic engineering. While researchers have been able to stably genetically transform Picea conifers, Pinus conifersxe2x80x94and particularly Southern yellow pinesxe2x80x94have proven to be extremely resistive to such modifications. Indeed, the relative ease of genetic transformation of Picea conifers in comparison to Pinus conifers is evident when examining the reports in the literature describing the success of Picea transformation in somatic embryogenic systems and the paucity of such reports for Southern yellow pines.
Although researchers have been able to routinely attain stable particle-mediated genetic transformations in Picea conifers, historically almost all of such transformations reported in Pinus conifers, particularly Southern yellow pines, have, upon examination, been found to be transient transformations or transformation of tissue which was not subsequently regenerable into whole plants. While transient, or non-integrative, transformation can be achieved easily in many tissues and stages of Pinus embryo development, the ability to achieve stable transformation in a tissue capable of producing whole plants is the key to a successful gene transfer system.
It has been found that a successful stable genetic transformation protocol is heavily dependant on the employment of an efficient tissue culture system. Moreover, efficient tissue culture methods must be coordinated with gene transfer at a receptive stage of embryo development in order to achieve stable genetic transformations. The stage of development at which transformation has been carried out in order to attain the regeneration of transgenic plants in Picea conifers (i.e. in embryogenic tissues initiated from cotyledonary embryos) does not give rise to embryogenic tissues capable of regenerating whole plants in conifers of the genus Pinus. Furthermore, in Picea conifers embryogenic tissues initiated from earlier stage embryos, such as pre-stage 3 somatic embryos, have not given rise to transformed plants. Indeed, the methods taught by Ellis in U.S. Pat. No. 5,681,730 for obtaining and genetically transforming somatic embryos in Picea conifers have not been found effective when applied to Pinus conifers. However, in the genus Pinus we have found that transformation steps can be successfully combined with a tissue culture system to derive embryogenic cultures from pre-stage 3 somatic embryos, pre-stage 3 zygotic embryos or somatic embryogenic tissue containing pre-stage 3 somatic embryos which are capable of regenerating whole transgenic plants.
It has long been known to those skilled in the art of transformation that a brief osmotic treatment at the time of transformation will increase transient expression of the transgene. In conifers, a treatment with elevated levels of inositol has been shown to be of benefit in transformation of white spruce, Picea glauca (Clapham et al. 1995). However, in Pinus taeda and P. taedaxc3x97P. rigida hybrids, the same treatment with elevated levels of inositol (i.e., levels greater than about 0.2 M) is detrimental to both growth and embryo development. In Pinus radiata, a pretreatment with sorbitol increased transient expression of a transgene (Walter et al. 1994), but such a pre-treatment has not been taught for obtaining stable expression and regeneration of transformed pine plants (Walter et al. 1997), perhaps because such treatments can also be detrimental to the regeneration of pine plants. To address these problems in the genus Pinus, we have developed a variety of preparation media for use before transformation and selection in pines. The use of the preparation media facilitated the recovery and development of stable genetically transformed embryos.
Therefore, an object of the present invention is to provide a method of genetically engineering plants of the genus Pinus and Pinus interspecies hybrids.
Another object of the present invention is to provide a method for stably transforming embryogenic tissues of the genus Pinus and Pinus interspecies hybrids.
A further object of the invention is to produce stably transformed embryos of the genus Pinus and Pinus interspecies hybrids capable of further development into transgenic plants.
Yet another object of the present invention is to produce genetically engineered plants of the genus Pinus and Pinus interspecies hybrids.
The above objectives are achieved via the use of a particle-mediated genetic transformation method which employs embryogenic tissues from plants of the genus Pinus and Pinus interspecies hybrids. This method involves the use of particle-mediated gene transfer with embryogenic tissues which are in a particular stage of development, namely pre-stage 3 somatic embryos, pre-stage 3 zygotic embryos, or somatic embryogenic tissue containing pre-stage 3 somatic embryos. It is preferred to accomplish this by employing a multi-step method which: a) prepares pre-stage 3 (i.e., pre-cotyledonary) somatic embryos, pre-stage 3 zygotic embryos, and/or somatic embryogenic tissue containing pre-stage 3 somatic embryos as the receptive target tissue for gene transfer via culturing the target tissue on preparation media, b) employs particle-mediated gene transfer to insert DNA into the target tissue, and c) exposes the bombarded tissue to selection media in order to identify and develop transformed embryogenic lines. Where desired, additional steps can be utilized to both cryopreserve such lines and to develop the transformed embryogenic lines into plants.
This method results in recovery of transgenic events through all stages of the transformation process leading to the production of transgenic pine trees (even historically recalcitrant Southern pines). This method also allows the transfer of genetic material to embryogenic cultures that may be used to establish clonal plantations of pine trees that are improved economically through expression of the transferred genetic material. This method further permits the development of transgenic embryos from embryogenic tissue which has been cryopreserved.
The invention also encompasses the genetically transformed embryos produced via the method and the transgenic plants derived therefrom.